An efficacious and safe vaccine is needed for Tuberculosis. The goal of this project is to develop a novel TB vaccine candidate strains of M. tuberculosis and M. smegmatis. Safety is achieved by generating attenuated mutants that fail to grow in immunocompromised mice. To generate a more efficacious TB vaccine, this project will generate mutants that have enhanced immunogenicity. Enhanced immunogenicity can be achieved by identifying mutations that inactivate determinants that evade host innate and adaptive immune responses. M. tuberculosis has the ability to actively suppress the induction of THI cytokines. Aim 1 utilizes a screen employing a macrophage cell line containing the gene encoding the green fluorescent protein fused to the IL-12 inducible promoter. Transposon and null deletion mutants of attenuated M. tuberculosis will be screened for the ability to induce IL-12. M. tuberculosis actively prevents apoptosis. Aim 2 uses a genetic screen to identify apoptotic blocking effectors (abe) mutations. The mutants isolated in both Aim 1 and Aim 2 will be evaluated for their efficacy in protecting mice against aerosolized M. tuberculosis challenges using both M. tuberculosis H37Rv and the KZN strain from South Africa. Preliminary data for Aim 3 has identified a novel set of genes in M. smegmatis named ike for innate killing evasion. The M. smegmatis Aike deletion Mice immunized with M. smegmatis Aike mutant containing a set of M. tuberculosis genes (named IKEPLUS) elicits a bactericidal immunity against M. tuberculosis. Heterologous prime and boosts with ILEPLUS and attenuated M. tuberculosis will be explored.